Multichannel bioluminescent sensors

ABSTRACT

Two or more strains of bioluminescent micro-organisms are used with multichannel luminescence sensors and logic circuitry to enable detection of specific chemical vapors. The organism strains are selected to produce predictable light-output variations when exposed to the specific vapors of interest, and signals analogous to light-output variations are combined in the logic circuitry to generate an alarm signal or other output when the specific vapors are sensed. Light-variation signals not conforming to the predicted pattern are rejected by the logic circuit to suppress an alarm or output when the organisms are exposed to vapors not of interest. Circuitry used with the system enables use of conventional photocells at very low light levels, and provides automatic compensation for long-term drift in organism light output.

[451 Nov. 19, 1974 MULTICHANNEL BIOLUMINESCENT SENSORS Inventors: Roy R.Sakaide, Woodland Hills; Clifford A. Shank, Canoga Park, both of Calif.

Bausch & Lomb Incorporated, Rochester, NY.

Filed: Sept. 27, 1973 Appl. No.: 401,287

[73] Assignee:

209; 23/230 B, 254 E, 232 E; 340/237 R, 242

[56] References Cited UNITED STATES PATENTS Jordan et al. 356/218 X Webb356/246 X Ede 250/209 X Leaf 250/458 2/1968 7/1970 l/l97l 3/19723,797,999 3/1974 Witz et al 23/230 B X Primary Examiner-Walter StolweinAttorney, Agent, or Firm-Frank C. Parker; Bernard D. Bogdon [57]ABSTRACT Two or more strains of bioluminescent microorganisms are usedwith multichannel luminescence sensors and logic circuitry to enabledetection of specific chemical vapors. The organism strains are selectedto produce predictable light-output variations when exposed to thespecific vapors of interest, and signals analogous to light-outputvariations are combined in the logic circuitry to generate an alarmsignal or other output when the specific vapors are sensed.Light-variation signals not conforming to the predicted pattern arerejected by the logic circuit to suppress an alarm or output when theorganisms are exposed to vapors not of interest. Circuitry used with thesystem enables use of conventional photocells at very low light levels,and provides automatic compensation for long-term drift in organismlight output.

8 Claims, 3 Drawing Figures PATENTL m 1 919m sum 30? 3 MULTICHANNELBIOLUMINESCENT SENSORS BACKGROUND OF THE INVENTION The use ofbioluminescent micro-organisms (such as bacteria or fungi) as sensorsfor toxic materials in gaseous (including aerosol) form is described inUS. Pat. No. 3,370,175. The use of these organisms is not limited todetection of toxic materials, and a variety of gases not usuallyconsidered toxic will produce a change in bioluminescence which can-bemeasured and used to generate an output signal or activate an alarm. Anygiven strain of organisms may, however, respond with a light-outputchange upon exposure to a number of different materials, and falseoutputs may be generated by a detection system which is intended torespond to the presence of only a specific material.

We have found that each bioluminescent organism strain has its ownpredictable and repeatable response characteristics upon exposure to anygiven gas. Broadly speaking, the light output upon such exposure mayinitially increase, decrease, or remain unchanged. When an increase ordecrease occurs, finer distinctions may sometimes be noted which canenhance the selectiveresponse properties of the strain. For example, the

magnitude of the change, the time required to return to a stable levelof luminescence, and the waveform of the variation are allcharacteristics which may aid in identifying the presence of a specificgas of interest.

Two or more organism strains are selected to produce patterns oflight-output variations which occur only in the presence of specificmaterials of interest. For example, strain X may respond positively(increased light output) to gases A, B, C and D, and have no response ora negative response to other gases. Strain Y may respond negatively(decreased light output) to gas A and certain other gases, but respondpositively (or not at all) to gases B, D and D. A logic circuit set torecognize the combination of a positive strain-X response and a negativestrain-Y response will generate the desired output or alarm only whenthe strains are exposed to gas A.

In accordance with the invention, the luminescence or emitted light ofthe several strains is sensed with photocells or similar transducers togenerate electrical signals analogous to organism light output. Thesesignals, suitably amplified, are combined in a logic circuit whichgenerates an output signal only when a predetermined pattern ofvariations occurs. The use of multiple sensors greatly reduces the riskof an undesired alarm upon exposure of the organisms to gases not ofinterest, and substantially limits alarms to those cases where apreselected gas of interest is detected.

The circuits here disclosed permit use of simple photo-cells (ratherthan more complex and expensive photomultipliers or other sensors) tomonitor luminescence, while preserving adequate sensitivity to detectminute variations about an initially low level of light output. Thesystem is thus useful to detect trace quantities of gases which producerelatively small variations in the average light output of theorganisms. Amplifiers used with the photocells include automatic gaincontrol to compensate for long-term variations in average light outputas the organisms progress through their lift cycles.

SUMMARY OF THE INVENTION Briefly stated, the system of this invention isarranged for detection of a specific vapor in an atmosphere beingsampled, and includes a plurality of bioluminescent sensors usingdifferent strains of micro-organisms having predetermined light-outputcharacteristics upon exposure to the vapor. A transducer means,preferably a plurality of photo-resistive cells, is disposed to receivelight from the sensors and generate electrical signals analogous to theluminescence of each sensor. A circuit means is connected to thetransducer means to receive the transducer signals, and to generate anoutput signal for activating an alarm device when the transducer signalsrepresent the predetermined luminescence characteristics arising fromexposure of the micro-organisms to the specific vapor.

BRIEF DESCRIPTION OF THE DRAWINGS DESCRIPTION OF THE PREFERREDEMBODIMENT FIG. 1 is a block diagram of a multi-channel bioluminescentsensor system 10 according to the invention.

The system is shown in a two-channel configuration, but a larger numberof channels may be used if the material to be detected requires morethan two strains of bioluminescent organisms to insure an appropriatelyselective response of the instrument.

System 10 includes a sample manifold 12 having a pump 13 at its exhaustend. A central part of the manifold is divided into two parallel lines12a and 12b each connected in series with a respective light-tightexposure chamber 14a and 14b. A housing or cartridge 15 is removablymounted in each exposure chamber, and supports a body of nutrientmaterial (such as conventional agar) and bioluminescent micro-organisms.Light emitted by the organisms is sensed by a photosensitive transducer16 mounted in each chamber 14. The transducer is preferably acadmium-sulfide photoresistive cell.

A gas sample to be monitored for presence of a specific material isadmitted to the inlet of manifold 12, and drawn through the manifold andexposure chambers by pump 13. Preferably, a humidifier 17 is inserted inseries with the manifold inlet line (or within the exposure chambers) toinsure that the gas being sampled is sufficiently humid to avoid dryingof the organisms and nutrient material. The manifold and exposurechambers should preferably be maintained at a rela-j tively constanttemperature, and these assemblies may be mounted in aconstant-temperature enclosure (not shown) if the instrument is used ina varying temperature environment.

Each photosensitive transducer 16 is connected to a photocell amplifier20 having an automatic-gaincontrol circuit to compensate for long-termvariations in average light level as the organisms progress throughtheir life cycles. As will be explained in greater detail below,photo-cell amplifier 20 includes a pair of capacitively coupledoperational amplifiers, the photosensitive transducer acting as avariable resistance in the feedback loop around the first-stageamplifier. The output of amplifier 20 is fed to a unity-gain phaseinverter 22, and also directly to a circuit 23 which provides polarityselection and adjustment of an alarm set point. Output of phase inverter22 is also connected to circuit 23, so the output signal of amplifier20may be fed to circuit 23 in either true polarity or inverted polarity.

The output signals from the several circuits 23 in the channels of thesystem are then fed to a mode-selection circuit 25 which drives a lamp,warning buzzer, or other alarm device. The mode-selection circuitincludes switching and logical circuitry enabling the operator toestablish a particular variation of light output variations from theorganisms which will produce an alarm output.

Referring now to the detailed schematic diagram in FIGS. 2A and 28, aresistor R1 and zener diode D1 are connected in series between apositive power input terminal 30 and a ground terminal 31. A batterypower source (not shown) of about 15 volts is suitable for operating theinstrument. Zener diode D1 establishes a constant potential of about 2.4volts at a junction 32 of the diode and resistor R1.

Photocell amplifier 20 includes an operational amplifier Al having afirst input terminal 26a connected to junction 32 through an inputresistor R2. A second input terminal 26b of the operational amplifier isconnected to ground. The operational amplifier has an output terminal27. An automatic gain control (AGC) circuit 28 is connected in serieswith photo-sensitive transducer 16 to form a feedback loop betweenoutput terminal 27 and first input terminal 26a of the opera tionalamplifier.

The AGC circuit includes a capacitor C1 and a potentiometer R4 (asensitivity matching control for the several channels) connected inparallel between output terminal 27 and ground. The arm of thepotentiometer is connected to one end of a diode circuit having two setsof series connected diodes D2 connected in backto-back relationship. Theother terminal of the diode circuit is coupled through a capacitor C2 toground, and is also connected to the gate electrode of a fieldeffecttransistor T1. One of the conductive electrodes of transistor T1 isconnected to ground, and the other electrode to the junction oftransducer 16 and a resistor R which is in turn connected to amplifieroutput terminal 27.

Photocell amplifier 20 further includes a low-pass AC amplifier 29 witha frequency range of about 0.05 to 2.0 HERTZ. Amplifier 29 includes anoperational amplifier A2 having a first input terminal 30 connectedthrough a resistor R7 and a pair of series-connected capacitors C4 andC5 to output terminal 27. Amplifier A2 has a second input terminal 31connected to ground, and also to first input terminal 30 throughseries-connected resistors R8 and R9. A pair of paralleled back-to-backconnected diodes D3 (effecting rapid charging of capacitors C4 and C5during large signal operation) are connected to the junction ofcapacitor C5 and resistor R7, and to the junction of resistors R8 andR9. A capacitor C6 and resistor R10 are connected in parallel betweenfirst input terminal 30 and an output terminal 33 of amplifier A2.

Phase inverter 22 includes an operational amplifier A3 having a firstinput terminal 36 connected through an input resistor R12 to outputterminal 33 of the lowpass AC amplifier. A second input terminal 37 ofamplifier A3 is grounded. A feedback resistor R13 is connected betweenan output terminal 38 and first input terminal 36 of amplifier A3.

A three-position polarity-selection switch 41 has a positive terminalconnected to output terminal 38, an open-circuit off terminal, and anegative terminal connected to output terminal 33. An arm 42 of switch41 is connected to mode-selection circuit 25 as described below.

A recorder and meter circuit 45 includes a pair of paralleledback-to-back connected diodes D4 connected between terminal 38 and arecorder output terminal 46. A single-pole single-throw shorting switch47 is connected across diodes D4. A meter selector switch 49 has a firstterminal connected to the positive power source of the circuit through aresistor R15. Two other terminals of the switch are connected torecorder output terminal 46 through respective ranging resistors R16 andR17. The arm of switch 49 is connected to a meter output terminal 50.

System 10 of this invention uses at least two channels of the type justdescribed, and a second channel is schematically indicated at the lowerpart of FIG. 2A. There may be three or more channels in an instrumentusing three different strains of organisms for highly selectivedetection of a particular material. These additional channels arepreferably identical to the circuit described above.

Referring to FIG. 2B which is a continuation of FIG. 2A, mode-selectioncircuit 25 includes a pair of identical trigger circuits 52 each havingan operational amplifier A4 arranged to act as a binary or two-stateswitch. Each amplifier has a first input terminal 53 connected throughan input resistor R18 to a junction 54 of a series-connected resistorR19 and zener diode D5 coupled between the positive power input terminaland ground. A second input terminal 55 is connected through a resistorR20 to a respective switch arm 42 at the output of phase inverters 22.Terminal 53 of each operational amplifier is also connected through analarm set-point rheostat R21 to ground.

An output terminal 58 of each amplifier A4 is connected through aseries-connected diode D6 and resistor R22 to the base electrode ofatransistor l2. The collectors of the two transistors are connected tothe positive power input terminal, and the emitters of the transistorsare connected together through a pair of seriesconnected resistors R23.

Circuit 25 also includes an operational amplifier A5 which acts as anand gate to generate an output signal when receiving inputs from both ofthe trigger circuits just described. Amplifier A5 has a first inputterminal 60 connected through a resistor R24 to junction 54, and asecond input terminal 61 connected through a resistor R25 to a junction63 of the two resistors R23. Junction 63 is also connected to groundthrough a level-setting rheostat R25A.

An output terminal 65 of amplifier A5 is connected to the base electrodeof a transistor T3 through a seriesconnected diode D7 and resistor R26.The collector electrode of transistor T3 is connected to the positiveinput power terminal.

A mode selector switch 66 has a first terminal 67 connected to theemitter of transistor T2 (channel A), and a second terminal 68 connectedto the emitter of transistor T3. A third terminal 69 is connected to theanode of a diode D8 having a cathode electrode connected to a fourthterminal 70 of switch 66. Another diode D9 has its anode connected toterminal 67 and its cathode connected to terminal 70. Terminal 70 isalso connected to the emitter of transistor T2 in the trigger circuitassociated with the second channel (channel A) of the system.

Connected between the emitter electrode of each transistor T2 and groundis a series-connected resistor R27 and light-emitting diode D10. Anotherlight emitting diode D11 is connected between ground and the arm ofswitch 66 through a resistor R28. A potentiometer R29 is connectedbetween the arm of switch 66 and ground. A warning horn or similar audioalarm 73 is connected between the arm of potentiometer R29 and ground.

Component types and typical values for the system just described are asfollows:

R1 8.2K ohms R2 approx. 1-5 megohms, depending on photocell R3, 10, 20,25 22 megohms R4, 5,7, 8, ll, l4, 15, 18,24 -100K ohms R6 approx. l-lmegohms, depending on photocell R9, 17, 22, 26 5.1K ohms R12, 13 47Kohms R16 120K ohms R19, 23, 29 10K ohms R21 25K ohms R25A 20K ohms R271.3K ohms R28 600 ohms C1 0.1 mfd.

C2 2 mfd.

C3 0.001 mfd.

C4, 5 22 mfd.

C6 0.0l mfd.

Tl 2N5,486

T2 2N4,40O

The operation of system 10 will now be described in terms of atwo-channel system having channels A and B as shown in the drawings. Theamplifiers and other components in each channel provide a D-C voltagelevel of several volts which is proportional to the light output of anassociated body of bioluminescent organisms in a respective cartridge15. These organisms typically have a luminosity level in the range of0.01 to 0.0001 foot candles (approximately 10' to 10 watts percentimeter The circuit is capable of linearly detecting changes of about0.1 percent to 5.0 percent from the average steady-state light output ofthe organisms, while maintaining a signal-to-noise ratio of about 100 tol for a signal stemming from a 1 percent intensity change.

Photo-sensitive transducer 16 is preferably a cadmium-sulfidephoto-resistive cell which is capable of detecting these low-level lightoutputs, and of providing acceptable signals for further processing andamplification by the electronic circuitry. The signal from thephoto-resistive cell is conditioned by the circuitry just described toprovide desired output levels, while maintaining an acceptablesignal-to-noise ratio, linearity, 65

AGC circuit 28 provides a dual-mode time constant, and uses field-effecttransistor T1 as a variable resistance to control the amount of feedbacknecessary to maintain constant output from amplifier A1. This dualmodetime constant is provided by series-parallel backto-back diodes D2 whichact as a voltage-dependent resistance of an RC time-constant networkdriving the gate of the field-effect transistor. At detector balance,the voltage across diodes D2 is essentially zero, and the resulting highresistance of the diodes effects a long time constant for the circuit.

Short-term signals resulting from slight light-output changes of theorganisms are thus transmitted directly to the output of the circuitwithout being appreciably affected by the AGC circuit. Cumulativesignals resulting from long-term drift in organism light output (or frompower turn-on or other large transient conditions) continually reset theAGC circuit to constant operating conditions without requiring anundesirable long stabilization time for the circuit.

The input to each amplifier A1 is a D-C reference signal applied throughresistor R2. Gain of this amplifier is established by the ratio of thecombined resistance of transducer 16 and calibrating resistor R6 to theresis tance of input resistor R2. This arrangement is useful as itcompensates for the proportionate response time characteristic ofcadmium-sulfide photocells to various light levels.

With a constant-gain amplifier, the result of this slowresponsecharacteristic would be smaller signals as organism luminosity decreasedduring the later stages of organism life. The circuit herein disclosed,however, includes the photocell resistance (which is inverselyproportional to incident light) as part of the operational-amplifierfeedback gain-determining resistance. The circuit thus increasesoperational-amplifier gain with lower organism luminosity, therebymaintaining relatively constant output signals. By compensating for therecognized characteristics of cadmium-sulfide photocells, the circuitthus responds uniformly to bioluminescent organisms having a wide rangeof ambient or steady-state luminosities.

Switches 41 and 66 provide a logical arrangement capable of detectingtwelve different states of organism luminosity outputs. That is, analarm can be generated for any of the following specific conditions ofchannels A and B.

Plus A Alone Plus A or Plus B Plus A and Plus B Minus A Alone Plus A orMinus B Plus A and Minus B Plus B Alone Minus A or Plus B Minus B andPlus B Minus B Alone Minus A or Minus B Minus B and Minus B RheostatsR21 set the triggering point of each channel, and the output state ofeachchannel is independently indicated by light-emitting diodes D10. Thetotal system output received from mode-selector switch 66 o is indicatedon light-emitting diode D11 and by audio alarm 73. Meter and recorderoutputs for each channel are provided by circuits 45, and switch 47 isused as a noise compression adjustment.

What is claimed is:

1. A system for detecting presence of a specific vapor in an atmospherebeing sampled, comprising:

a plurality of bioluminescent sensors utilizing different strains ofmicro-organisms which have predetermined luminescence characteristicsupon exposure to the vapor;

transducer means for monitoring the sensors and generating a pluralityof signals representing luminescence of each sensor;

circuit means connected to the transducer means to receive thetransducer signals and to generate an output signal when the transducersignals represent the predetermined luminescence characteristicsproduced by exposure of the micro-organisms to the specific vapor.

2. The system defined in claim 1 wherein the circuit means comprises anamplifier for each transducer means, a mode-selection means for sensingpredetermined and selectable combinations of outputs from the amplifiersand generating an alarm output upon sensing a selected combination,indicating means connected to the mode-selection means to receive thealarm output, and coupling means connected between the amplifiers andmode-selection means.

3. The system defined in claim 2 wherein the coupling means comprises aphase inverter for each amplifier connected to receive an output fromthe amplifier, polarity-selection means connected to the modeselectionmeans, and means connecting the amplifiers, polarity-selection means andmode-selection means whereby the output from each amplifier is deliveredto the mode-selection means in a selectable polarity.

4. The system defined in claim 3 wherein the modeselection means isarranged to generate an alarm output upon receiving an amplifier outputof predetermined polarity, and also upon receiving selected combinationsof several such amplifier outputs.

5. The system defined in claim 4 wherein transducer means comprises aphoto-resistive cell for each bioluminescent sensor, each cell beingconnected in circuit with a respective amplifier.

6. The system defined in claim 5 wherein each amplifier has input andoutput terminals, and the photoresistive cell is connected between theterminals to constitute a portion of a feedback loop for the amplifier.

7. The system defined in claim 5 wherein each amplifier has input andoutput terminals, and each amplifier includes an automatic-gain-controlcircuit connected in series with the associated photo-resistive cellbetween the terminals in feedback relationship.

8. The system defined in claim 7 wherein the automatic-gain-controlcircuit is arranged to adjust amplifier gain to compensate for long-termvariations in steady-state luminescence emitted by the associatedmicro-organisms.

'UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 9,53 Dated N v 19, 197 1 In en PQY R. Sakaida et al.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Page 1, in both places change the inventor s name from "Sakaide" to--Sakaida--.

Col. 1, line 6 delete "lift" and substitute therefor -life--.

Signed and Scaled this thirteenth Day of April 1976 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN Alresn'ng Officer (ummissimzer of Patentsand Trademarks UNITE STATES PATENT OFFICE.

CERTIFICATE OF CORECTION Patent No. 3 49, 53 Dated November 9, 97

Izjlventofls) iaRoy R. Sakaida g Clifford A. Shank It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Col. R, line 43, after trans:Lscor" delete "i2" and substitute therefor--T2--.

Signed and sealed this 18th day ofFebruary 1975.

(SEAL) Attest: I V

C. MARSHALL DANN RUTH C. MASON Conunissioner ofv Patents ArrestingOfficer and Trademarks FORM PO-105O (10-69) USCOMWDC 6O376 P69 U-S.GOVERNMENT PRINTING OFFICE I959 O 356-334

1. A system for detecting presence of a specific vapor in an atmospherebeing sampled, comprising: a plurality of bioluminescent sensorsutilizing different strains of micro-organisms which have predeterminedluminescence characteristics upon exposure to the vapor; transducermeans for monitoring the sensors and generating a plurality of signalsrepresenting luminescence of each sensor; circuit means connected to thetransducer means to receive the transducer signals and to generate anoutput signal when the transducer signals represent the predeterminedluminescence characteristics produced by exposure of the micro-organismsto the specific vapor.
 2. The system defined in claim 1 wherein thecircuit means comprises an amplifier for each transducer means, amode-selection means for sensing predetermined and selectablecombinations of outputs from the amplifiers and generating an alarmoutput upon sensing a selected combination, indicating means connectedto the mode-selection means to receive the alarm output, and couplingmeans connected between the amplifiers and mode-selection means.
 3. Thesystem defined in claim 2 wherein the coupling means comprises a phaseinverter for each amplifier connected to receive an output from theamplifier, polarity-selection means connected to the mode-selectionmeans, and means connecting the amplifiers, polarity-selection means andmode-selection means whereby the output from each amplifier is deliveredto the mode-selection means in a selectable polarity.
 4. The systemdefined in claim 3 wherein the mode-selection means is arranged togenerate an alarm output upon receiving an amplifier output ofpredetermined polarity, and also upon receiving selected combinations ofseveral such amplifier outputs.
 5. The system defined in claim 4 whereintransducer means comprises a photo-resistive cell for eachbioluminescent sensor, each cell being connected in circuit with arespective amplifier.
 6. The system defined in claim 5 wherein eachamplifier has input and output terminals, and the photo-resistive cellis connected between the terminals to constitute a portion of a feedbackloop for the amplifier.
 7. The system defined in claim 5 wherein eachamplifier has input and output terminals, and each amplifier includes anautomatic-gain-control circuit connected in series with the associatedphoto-resistive cell between the terminals in feedback relationship. 8.The system defined in claim 7 wherein the automatic-gain-control circuitis arranged to adjust amplifier gain to compensate for long-termvariations in steady-state luminescence emitted by the associatedmicro-organisms.